M. tuberculosis (Mtb) is a global health problem and bioterrorism threat but little new chemotherapy against Mtb has emerged in decades. A fresh approach to chemotherapy is to target enzymes the pathogen needs to survive in the metabolic niche it occupies in the host, and/or enzymes that the pathogen needs to resist the stresses that are imposed on it by the host immune response. The metabolism of Mtb in the host depends on oxidation of fatty acids as a carbon source and synthesis of fatty acids as precursors of cell wall lipids. An early stage of fatty acid synthesis is the formation of acetyl-coenzyme A (acetyI-CoA) and other very short-chain acyl CoA's, chiefly from pyruvate dehydrogenase (PDH) and branched chain keto-acid dehydrogenase (BCKADH). A major host defense against Mtb is the imposition of oxidative/nitrosative stress. Remarkably, Mtb appears to rely on one enzyme as a shared component of PDH and BCKADH, and this enzyme also helps to protect the bacillus against oxidative/nitrosative stress: Rv2215 or dihydrolipoamide acyltransferase (DLAT) (formerly "SucB"). Thus, inhibitors of DLAT may cripple Mtb at multiple levels: by interfering with operation of the TCA cycle for generation of ATP and precursors of heme and amino acids; by interfering with synthesis of acetyl CoA, propionyl CoA and acetoacetyl CoA for the glyoxalate shunt and synthesis of fatty acids; and by interfering with antioxidant defense. We have screened a combinatorial chemical library and identified relatively potent and specific inhibitors of Mtb's DLAT. These compounds kill M. bovis BCG and Mtb in culture while sparing homologous host enzymes and macrophages. The goal of this application is to use these findings as a rationale, and these inhibitors as a starting point, to develop a compound for testing in mice as a potential therapeutic for tuberculosis with a new mechanism of action.